Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus. CNTs impair neuronal exocytosis through specific proteolysis of essential proteins called SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptor). This proteolysis prevents the release of neurotransmitter at neuromuscular junctions, resulting in muscle paralysis. The identification of the means by which a CNT properly identifies and cleaves its target SNARE is therefore of clinical and biological significance.
Site-specific SNARE hydrolysis is catalysed by the CNT light chains, a unique group of zinc-dependent endopeptidases. The structures of three CNT light chains suggest that substrate recognition cannot occur at the active sites of these proteases, because the catalytic pocket composition and geometries of BoNTs A, B, and E are essentially identical. Furthermore (and atypically for endopeptidases), light-chain activity can be strongly influenced by remote substitutions and deletions. for example, conserved motifs containing acidic residues in the substrates were shown to be required for normal levels of light-chain activity and led to the proposal that the light chains may use exosites for efficient substrate recognition and cleavage. However, structural data concerning the locations and functions of these exosites have remained elusive. Accordingly, there is a need in the art to determine the precise structure and amino acids involved in the binding of CNT light chains to exosites on the target SNAREs.